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Ouyang et al. Stem Cell Research & Therapy (2018) 9:246 Page 6 of 12

Fig. 3 Intracavernous pressure (ICP) during cavernous nerve (CN) electrostimulation at 4 weeks after surgery. a-d Representative ICP responses for the
sham group, CNI rats stimulated 4 weeks after IC injection of PBS, MSCs, and MSC-Exos. The colored bar denotes the 50 s CN electrical stimulation. e, f
The ratios of the total ICP (area under the curve) and maximal ICP to MAP are recorded. Each bar depicts the mean ± standard deviation from n =8
animals per group. *p < 0.01 compared with the PBS vehicle group

(MSCs, 0.65 ± 0.03, 0.69 ± 0.03; MSC-Exos, 0.50 ± 0.03, significant decrease in smooth muscle content in the
0.60 ± 0.04) compared to the PBS group (p <0.01). corpus cavernosum of the PBS group compared to the
sham group (Fig. 5a). The MSCs and MSC-Exos treat-
Transplantation of MSC-Exos increase penile nNOS ment groups exhibited partial but significant restoration
expression and alleviate cell apoptosis of smooth muscle content after CNI, as shown by west-
The expression of nNOS in the corpus cavernosum was ern blot analysis (Fig. 5c, d). The corpus cavernosum
detected by immunofluorescent staining 4 weeks after was evaluated for the smooth muscle/collagen ratios on
injection with MSCs or MSC-Exos. The data revealed slides stained with Masson’s trichrome (Fig. 5b). In the
that nNOS expression was significantly higher in the sham group, the smooth muscle/collagen ratio was
sham (0.43 ± 0.04), MSCs (0.33 ± 0.04), and MSC-Exos 0.166 ± 0.013. In the penile tissue from rats from that re-
(0.33 ± 0.10) groups than in the PBS group (0.18 ± 0.03; ceived MSCs or MSC-Exos, the smooth muscle/collagen
p < 0.01) (Fig. 4a, b, c). TUNEL assays were applied to ratio (0.118 ± 0.013 or 0.107 ± 0.013) was almost fully
assess cell apoptosis in the corpus cavernosum in vivo. preserved and significantly higher than in the PBS group
In vivo TUNEL assays confirmed that MSC-Exos effect- (0.054 ± 0.010) (Fig. 5e,*p < 0.01).
ively protected cells from apoptosis after CNI. Addition-
ally, MSC-Exos could alleviate the apoptosis of CCSMCs identification
nNOS-positive cells, which also contributed to the re- Fig. 6 shows primary cells that have migrated out from
covery of erectile function (Fig. 4a). Western blots were the corpora tissues after 4–7 days and achieved conflu-
performed to evaluate the expression of caspase-3 after ence after 2–3 weeks. It also shows passaged cells grow-
4 weeks of treatment with MSCs and MSC-Exos. The ing in a whirlpool-like pattern (Fig. 6a). Over 95% of the
expression of caspase-3 significantly decreased in the cultured cells were identified by immunofluorescence
MSCs (1.52 ± 0.07) and MSC-Exos (1.70 ± 0.06) groups (IF)-labeling of calponin (Fig. 6b), suggesting highly pure
compared to the PBS group (2.11 ± 0.15) (Fig. 4d, e). CCSMCs for the following experiments.

MSC-Exos treatment improves smooth muscle content Inhibition of CCSMCs apoptosis using exosomes
and the ratio of smooth muscle to collagen in the corpus To assess the anti-apoptotic effects of MSC-Exos on
cavernosum CCSMCs, we analyzed the viability of exosomes-
CNI caused corpus cavernosum smooth muscle atrophy, supplemented cells after H 2 O 2 treatment by flow cytome-
and computerized histomorphometric analysis showed a try. CCSMCs were cultured in the absence or presence of